A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

[Expression of DNA excision repair enzymes and lung cancer prognosis].

OBJECTIVE: To study the expression levels of three DNA damage excision repair enzymes: excision repair cross-complementing rodent repair deficiency gene 2 (ERCC2), uracil DNA glycosylase (UDG), and proliferating cell nuclear antigen (PCNA), in different lung tissues and their relationship with lung cancer prognosis. METHODS: The expression levels of ERCC2, UDG, and PCNA protein were detected using immunohistochemistry method. Cancer tissues and normal tissues adjacent to the cancer from 61 cases of lung carcinoma, and tissue samples from 18 cases of benign lung disease were studied. The relationship among ERCC2, UDG, and PCNA expression levels with lung cancer development and prognosis were studied using Ridit analysis method. RESULTS: The expression levels of all three proteins were not significantly different between cancer tissues and a normal tissues adjacent to the cancer (P > 0.05); but significantly different between cancer tissues and benign tissues, and between normal tissues adjacent to the cancer and benign tissues (P < 0.05). ERCC2 and UDG levels were higher in benign tissues than those in cancer and cancer adjacent tissues, while the PCNA level was the opposite. Only the UDG level in cancer adjacent normal tissues showed no significant difference compared to that of benign tissues (P > 0.05). There were no relationship among age, smoke, cancer metastasis, cancer type and ERCC2, UDG, PCNA expression levels. ERCC2 also showed no relationship with degree of malignancy and cancer size, while UDG and PCNA expression levels were correlated with cancer malignancy and cancer size (P < 0.05). Low UDG and high PCNA expression levels were correlated with higher malignancy and larger tumors. CONCLUSION: The expression levels of ERCC2, UDG and PCNA were significantly different in benign lung tissues, lung cancer tissues and lung cancer adjacent normal tissues. ERCC2 showed no significant relationship with lung cancer prognosis, while patients with low UDG and high PCNA expressions were more likely to have higher malignancy and larger tumors. UDG and PCNA were possible markers for evaluating lung cancer prognosis.

[Antisense ERCC1 RNA decreases the repair capability of damaged DNA in lung cancer cells induced by benzo[a]pyrene].

OBJECTIVE: To investigate the effect of ERCC1 gene on the repair capability of damaged DNA in lung cancer A549 cells induced by benzo[a]pyrene. METHODS: Recombinant plasmid expressing ERCC1 antisense RNA was constructed and transfected into A549 cells by Lipofectin reagent. The stable-transfected cell colonies were selected by hygromycin. Cell viability was determined by the MTT assay. The level of ERCC1 mRNA was measured by Northern Blot analysis. Single cell gel electrophoresis assay was applied to determine the cellular DNA damage and fifty cells for each group were counted. RESULTS: Seven positive colonies expressing ERCC1 antisense RNA were screened. There was no growth rate difference between the antisense-transfected cells and the parental cells. The endogenous mRNA level in transfected colonies decreased in varied degrees, i.e. 12% approximately 86% of that of the parental cells in Northern Blot assay. After 24 h treatment of 10 micro mol/l benzo[a]pyrene, the repair capability for DNA damage in transfected colonies was reduced to 29% approximately 71% of that of the parental cells. Also, a statistically significant correlation was observed between expression of ERCC1 mRNA and repair capability (r = 0.84). CONCLUSION: Antisense ERCC1 RNA decreased the repair capability for damaged DNA in lung cancer cells induced by benzo[a]pyrene.

[Study on the expression of DNA excision repair biomarkers in cispatin-treated lung cancer cell line].

OBJECTIVE: To study the expression levels of ERCC2, UDG, and PCNA in cisplatin-treated A549 cell line. METHOD: Comet assay, RT-PCR, and western blot were used to study the mRNA and protein expression levels of ERCC2, UDG, and PCNA. RESULTS: When treated with IC(20) cisplatin, the DNA damage level increased as the cisplatin treated time increased within 24 h of cisplatin treatment. The tail state 12 h and 24 h after treatment was 5.02 +/- 0.68 and 7.22 +/- 0.53 respectively, which was significantly higher than those of the controls (2.73 +/- 0.29). The tail state 24 h after treatment was not significantly different from that of the controls. The DNA damage level decreased to normal after cisplatin treatment in 24 h (tail state 3.64 +/- 0.7). The expression levels of ERCC2, UDG, PCNA protein (4.37 +/- 0.57, 5.47 +/- 0.46, 2.21 +/- 0.47 respectively) and mRNA (0.71 +/- 0.08, 0.74 +/- 0.06, 0.82 +/- 0.09) were increased significantly within 24 h exposure and decreased to normal 24 h after cisplatin treatment. The 3 enzymes' mRNA and protein expression increased when treated with cisplatin, but the changes of protein level were slower than those of mRNA levels. CONCLUSIONS: The DNA repair capability in A549 cells increases after cisplatin treatment. Cisplatin was a positive regulation of ERCC2, UDG, PCNA expression levels, which causes the increase of mRNA, and protein. The positive regulation only works in a short time and returns normal after 24 h of cisplatin treatment.

delta-Aminolevulinic acid dehydratase activity, urinary delta-aminolevulinic acid concentration and zinc protoporphyrin level among people with low level of lead exposure.

To evaluate the relationship of delta-aminolevulinic acid dehydratase (ALAD) activity, urinary delta-aminolevulinic acid (ALAU) level and blood zinc protoporphyrin (ZPP) concentration to low blood lead (PbB) levels, these biomarkers were determined for all subjects enrolled from a rural area of southeast China where people had low levels of exposure to lead. The mean values of PbB, ALAD, ALAU and ZPP were 67.11 microg/L (SD: 1.654, range: 10.90-514.04), 339.66 nmol ml(-1)h(-1) (1.419, 78.33-793.13), 20.64 microg/L (1.603, 2.00-326.00), and 0.14 micromol/L (3.437, 0.01-2.26), respectively. ALAD was inversely associated with low levels of PbB. ZPP was inversely related to low levels of PbB but positively related to relatively higher levels of PbB. Alcohol drinking contributed to low ALAD in men. Women had higher ZPP than men. ALAU had no significant association with PbB. In conclusion, ALAD possibly has a non-linear relation with low to moderate levels of PbB. At moderate levels of PbB, ZPP increases with increasing levels of PbB. ALAU is not suitable as an indicator for low levels of lead exposure.

[Gene polymorphism of myeloperoxidase and genetic susceptibility to lung cancer].

BACKGROUND & OBJECTIVE: A-463 bp G/A polymorphism is located in the promoter region of myeloperoxidase (MPO) gene was found to be associated with lung cancer susceptibility; however, this association in Chinese population remained unclear. The aim of this study was designed to explore this association in Chinese population. METHODS: MPO genotypes in 98 cases of primary lung cancer and 112 persons of healthy control were detected using PCR-restriction fragment length polymorphism assay(PCR-RFLP) in a case-control molecular epidemiology study. The association between this gene polymorphism and the risk of lung cancer in Chinese population was examined through comparing odds ratio(OR) and 95% confidence interval(CI) between two groups. RESULTS: In healthy control group, the frequencies of persons carrying G/G, G/A, and A/A genotypes were 47.3%, 42.9%, and 9.8%, respectively. In cancer group, the frequencies of the persons carrying above three genotypes were 63.3%, 33.7%, and 3.0%, respectively. There was no significant difference of the frequencies of heterozygote G/A between two groups (P >0.05). However, The A/A genotype was found significantly higher in cancer group than that in healthy control group(P< 0.025). The risk of lung cancer for person carrying at least one A allele was only 52% of the persons carrying G/G genotype (95% CI 0.29-0.93). In smoking population, allele A showed to be a significant protective factor (OR=0.41,P< 0.025), while such protective role was not observed in non-smoking group(P >0.25). CONCLUSION: MPO gene polymorphism was associated with susceptibility of lung cancer in Chinese population. The risk of lung cancer was decreased in the persons carrying allele A.

[Construction of nucleotide excision repair gene XPB antisense RNA expression plasmid and its functions].

BACKGROUND & OBJECTIVE: Nucleotide excision repair is an important pathway for cellular DNA damage repair. The drug resistance of tumor cell is often companied with the enhanced expression of DNA repair genes. Down-regulation of DNA repair capacity by antisense strategy can increase the drug sensitivity of tumor cells. The aim of this study was to construct the eukaryotic expression plasmid pcDNA-XPB/AS (XPB: xeroderma pigmentosum B) and to investigate the function of XPB gene and its roles in chemotherapeutic drug sensitivity in lung cancer A549 cell. METHODS: The XPB cDNA (69-520 bp) fragment amplified by reverse transcription polymerase chain reaction (RT-PCR) was inserted into pcDNA3.1/His plasmid with an inverted orientation. The recombinant plasmid was transiently transfected into A549 cells. The Adriamycin-induced DNA damage was compared between the transfected and the untransfected cells by single cell gel electrophoresis assay (SCGE). The cellular sensitivity to Adriamycin of the transfected and the untransfected cells was determined by MTT assay. RESULTS: The successful construction of antisense plasmid was proved by restriction map and sequence analysis. RT-PCR results showed that the XPB mRNA expression was inhibited in transfected A549 cells. SCGE showed that the cellular damage repair ability induced by 4.0 microg/ml Adriamycin was suppressed in transfected cells. MTT assay showed the sensitivity of the transfected cells to Adriamycin was different from the untransfected cells but without statistical meaning. CONCLUSION: The antisense plasmid constructed by the authors can down-regulate the expression of XPB mRNA in the transfected cells and inhibit the cellular DNA damage repair ability, providing a basis to further study the gene function of XPB.